The present invention relates in general to visual imaging of the ion distribution in tissue and, more particularly, to a method of producing visual images of ion distribution in human epidermis. The method of this invention can provide an extremely inexpensive and simple method to detect ion distribution compared with the prior art methods.
Studies suggest that ionic signals such as calcium and potassium play an important role in the homeostatic mechanism of the epidermal barrier function. Observation of these ions would provide important information to understand epidermal homeostasis. However, image analysis of ions in the skin is technically difficult. Previously, the distribution of calcium and potassium has been studied by electron microscopic analysis after calcium precipitation or by Particle Induced X-Ray Emission (PIXE) analysis of the skin. Although the results of these procedures provides important quantitative information, some other important elements such as hydrogen or magnesium, could not be observed by these methods because of their low atomic weight. Moreover, since these methods require dehydration or fixation of the sample without destroying the native chemical composition, they require complicated processes.
Secondary ion MS (SIMS)-based imaging technique also can be used for observation of intercellular elements. But this method also requires freeze-dry processing of the sample to be analyzed.
Although frozen hydrated and frozen freeze-dried sample preparations do provide optimal samples for SIMS analysis, neither approach preserves the living-state cell matrix. In frozen hydrated samples, the physical form of the cellular water is completely altered, and, in freeze-drying, the water is removed from the specimen. The choice between frozen hydrated and frozen freeze-dried sample preparations may depend on the type of SIMS analysis desired. The frozen hydrated analysis is preferred for static time-of-flight molecular imaging. Static SIMS experiments have also imaged molecules in tissue sections after air drying. On the other hand, frozen freeze-dried sample preparations are preferred in dynamic SIMS. Analysis of frozen hydrated cells under a dynamic primary beam preferentially removes water along the z-direction. This effect also enhances other analyte signals and causes false image contrasts in ion images. Additionally, frozen hydrated biological samples offer poor electrical conductivity, whereas conductivity is enhanced upon freeze-drying.
Further, freeze-drying may cause cell shrinkage and often damages some of the cellular morphology. To minimize this damage, sample freezing and freeze drying at low temperatures have been used. For example, morphological evaluations of fractured cells and membrane pieces prepared with a xe2x80x9csandwich fracturexe2x80x9d method using electron and laser scanning confocal microscopic techniques, revealed well-preserved membrane particles, mitochondria, lysosomes, Golgi apparatus, and cell cytoskeleton. This is not surprising, because the method quick freezes cell monolayers of xe2x89xa610 xcexcm thickness and freeze-dries the sample at temperatures of xe2x88x9280xc2x0 C. or lower.
The presence of external cell growth medium complicates the direct analysis of cultured cells by ion microscopy. Washing out the nutrient medium can expose calls to a foreign solution that may perturb them and affect their ionic composition. These obstacles are overcome by using a sandwich freeze fracture method developed in our laboratory.
It is therefore an object of the present invention to provide a method of imaging ions in tissue samples without any substantial alteration of the native chemical composition of the cell by an expensive and simple procedure.
A method is provided according to the present invention of producing a visual image of ions in tissue samples which comprises placing frozen thin tissue sample in contact with a thin membrane formed from a water soluble polymer gel or hydrophobic plastic film having incorporated in or placed thereon a fluorescent or color ion indicator which fluoresces or changes color under light of a particular wavelength. The thin frozen tissue sample is then subjected to light of a wavelength to cause the ion indicator to fluoresce or change color.
In a preferred method of the present invention, the tissue sample to be analyzed is a frozen tissue sample of human skin. The human skin sample can be any portion of the skin, but it is preferred that it is the epidermal layer and dermal layer, etc. The sample size is not limited and suitable sample size can be easily determined in any particular test. In a preferred embodiment the sample size is approximately 1 mmxc3x971 mm cross-section tissue sample. However, the size of the tissue sample may be dictated by the type of equipment used in photographing and irradiating the tissue sample. In one embodiment of the invention, a cross-section of the skin can be analyzed by cutting the skin sample with a cryostat to a thickness of about 10 xcexcm. Preferably, the frozen thin tissue sample is cut to a thickness of from about 5 to 10 xcexcm for use in a fluorescence microscope.
According to a preferred method of the present invention, the frozen thin tissue sample is brought into contact with a thin membrane of a water soluble polymer gel or hydrophobic plastic film. Preferably, the water soluble polymer used to form the plastic film is a polymer selected from the group consisting of polyacrylamide, polyvinyl alcohol, or polysaccharide which is gelated or solidified into sheet form by cross bridging or drying, or crosslinking or polymerization of a monomer.
When the membrane comprises a water soluble polymer gel such as polysaccharide, the membrane preferably comprises from about 2 wt % of said polymer and about 98% of water. The content of said polymer and water depends on the particular polymer being used to form the membrane. According to the present invention, the membrane upon which the tissue sample is placed forms a three-dimensional structure which prevents any substantial water flow inside the structure so as to prevent any substantial movement of an ion indicator in the membrane.
Incorporated in or placed in the thin membrane is a fluorescent or color ion probe which fluoresces or changes color under light of a particular wavelength. The concentration of the ion probe is not limited but is preferred to use about 10 xcexcg/ml based on the membrane being used.
In the process of the present invention for producing visual images of ions in tissue samples, any of the conventional ion probes can be used for the detection of any ions. For example, calcium ion probes can be used as described in a Japanese non-examined patent publication Tokkai HEI 2 -28542. Other suitable ion probes for calcium available from Molecular Probe of Eugene, Oreg. USA include Fura-2 (5-Oxazolecarboxylic acid, 2-(6-(bis(carboxymethyl)amino)-5-(2-(2-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, pentapotassium salt) Fluo-4. Preferred magnesium ion probes include Mag-Fura-2 (Oxazolecarboxylic acid, 2-[6-[bis(carboxymethyl)amino]-5-(carboxymethoxy)-2-benzofuranyl]-, tetrapotassium salt).
Preferred potassium ion probes include PBFI(1,3-Benzenedicarboxylic acid, 4,4xe2x80x2-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-,tetraanimonium salt). For example, sodium ion probes include SBFI(1,3-Benzenedicarboxylic acid, 4,4xe2x80x2-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-,tetraammonium salt). Also, zinc ion probes, for example, include TSQ(N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide). Suitable chloride ion probes, for example, include MEQ(6-methoxy-N-ethylquinolinium iodide). Suitable ion probes for reactive oxygen include, for example, carboxy -H2DCFDA (5-(and-6)-carboxy-2xe2x80x2,7xe2x80x2-dichlorodihydrofluorescein diacetate), and suitable lipid ion probes include, for example, Dil (1,1xe2x80x2-dioctadecyl-3,3,3xe2x80x2,3xe2x80x2-tetramethylindocarbocyanine perchiorate).
Among preferred probes are Calcium Green-1; AM ester (C-3011); Magnesium Green, AM ester (M-3735); PBFI, AM ester (P-1266); Sodium Green (S-6900); Newport Green (N-7990); Potassium probe PBFI; Chloride Probes 6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ, M-440), N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, E-3101), 6-Methoxy-N-ethylquinolinium iodide (MEQ, M-6886), and Lucigenin (L-6868); Membrane potential, JC-1 (T-3168), JC-9 (D-22421), and Dil, DiO, DiD,-General purpose fluorescent membrane stains, all of which can be obtained from Molecular Probes, Inc. of Eugene, Oreg., USA.
Preferred molecular probes for singlet oxygen include trans-1-(2xe2x80x2-methoxyvinyl)pyrene available from Molecular Probes of Eugene, Oreg. In addition, zinc probes for detecting Zn2+ include Newport Green, dipotassium salt, having the molecular formula C30H24Cl2K2N4O6. Suitable molecular probes for Fe2+, Cu2+ are Phen Green FL, dipotassium salt having the formula C33H18K2N4O5S; suitable indicators for probes for Mg2+ include Magnesium Green, pentapotassium salt, molecular formula C33H17Cl2K5N2O13; a preferred CA2+ probe is Calcium Green-1, hexapotassium salt having a molecular formula of C43H27Cl2K8N3O16and a preferred Na+ molecular probe is Sodium Green, tetra(tetramethylammonium)salt having a molecular formula C84H100Cl7N8O10, all of which are obtainable from Molecular Probes, Inc. of Eugene, Oreg.
The membrane for use in producing a visual image of ions in a tissue sample can be formed by first preparing an aqueous solution containing from about 5 to 20 wt % of polyacrylamide or polyvinyl alcohol or from about 0.05-2 wt % of polysaccharide together with an ion probe for the particular ion to be detected. A few drops of this aqueous solution can then be dripped on a glass slide which is preferably heated to a temperature of from about 40-50xc2x0 C. to vaporize a portion of the water. Alternatively, an agarose gel can be formed from an aqueous solution dripped onto a glass slide and then dried to form a thin gel membrane. In a preferred embodiment the polysaccharide is gelated or solidified into sheet form by cross-bridging or drying, or crosslinking or polymerization of a monomer on a flat surface such as a glass slide so as to form a very thin membrane. According to a preferred method, the ion indicator is incorporated into the material forming the membrane before it is cast into a thin film. However, the ion probe can also be applied as a surface coating on the gel membrane.
After the thin frozen tissue sample is placed in contact with the thin membrane, the sample is then subjected to light of a wavelength to cause the ion probe to fluoresce or change color and the color change or fluorescence of the ion probes is observed by means of an optical microscope, fluorescent microscope, confocal microscope, and/or multiphoton laser scanning microscope.